Upregulated PD-1 signaling antagonizes glomerular health in aged kidneys and disease

With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2). PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence-associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti–PD-1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti–PD-1 antibody treatment improved the health span of podocytes. Administering the same anti–PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS.


Immunostaining, Quantification and Visualization
Immunoperoxidase staining was performed on 4µm thick formalin fixed paraffin-embedded (FFPE) mouse and human kidney sections as previously described (89). Double immunostaining was performed on 4 µm thick frozen and FFPE sections as previously described (90). These immunohistochemical studies were approved by the University of Chicago institutional review board. The primary antibodies used in the study are summarized in Supplemental Table 2. To quantify immunohistochemistry, slides were scanned in brightfield with a 20x objective using a NanoZoomer Digital Pathology System (Hamamatsu City, Japan). The digital images were imported into Visiopharm software (Hoersholm, Denmark) and its Image Analysis Deep Learning module was trained to detect glomeruli and assess immunohistochemical staining-positivity for e.g., Collagen IV, p57 or ERG. The glomeruli ROIs were processed in batch mode generating per area outputs, cell counts and analyzed from 100% of the tissue sections. In the case of the parietal epithelial cells, images were collected using an EVOS FL Cell Imaging System (Life Technologies). Bowman's capsule length was measured by using ImageJ 1.46r software (National Institutes of Health) and the percentage of positivity were calculated by dividing it by Bowman's capsule length.
Two-dimensional images were detected on EVOS FL Cell imaging system (Thermo Fisher Scientific, Waltham, MA, USA) using a 20x objective and increased to a 200x magnification. Stained kidney sections were digitally imaged by the University of Washington Histology and Imaging Core (HIC) using a Hamamatsu whole slide scanner (Bridgewater, NJ, USA).

In situ Hybridization
RNA in situ hybridization was performed on the kidney tissues by following the manufacturer instructions for the RNAscope 2.0 FFPE Assay kit -BROWN (Advanced Cell Diagnostics). Briefly, kidneys were perfused and fixed in 10% formalin, dehydrated and embedded in paraffin. 4 µm paraffin sections were cut, dehydrated in 100% and 95% of Ethanol solutions. Sections were boiled at 100 0 C in EZprep buffer for 20 min followed by a protease incubation for 30 min at 37 0 C. Probes specific to mouse PDCD1 (Advanced Cell Diagnostics) were hybridized at 48 0 C for 2 hours in an oven, followed by a subsequent series of washing and signal amplification steps. Mouse specific positive and negative probes for PDCD1 gene were provided by Advanced Cell Diagnostics. Hybridization signals were detected by DAB staining. Stained tissues were imaged on an EVOS FL Cell Imaging System.

FLARE Staining and Expansion Microscopy
Hydrogel-expansion and FLARE staining of kidney sections were performed according to the published FLARE protocol (61) on 50 µm thick sections of cryo-preserved mouse kidney tissue. In brief, oxidized carbohydrates were labeled with ATTO 565 hydrazide, amines (proteins) were labeled with ATTO 647N NHS ester, and nuclei were labeled with the fluorescent DNA-binding dye SYBR Green I.
Expanded gels were transferred onto a poly-L lysine coated coverslips (24 mm by 50 mm, no. 1.5; Fisher Scientific, #12544E) and imaged immediately. Images were acquired using a Nikon A1R inverted pointscanning confocal microscope at the University of Washington Biology Imaging Facility. A CFI Apo LWD Lambda S 40× objective lens with 1.15 numerical aperture was used on the microscope. Threecolor 3D stacks were acquired with 100 nm lateral sampling and 200 nm axial sampling. A median-filter of 1 pixel radius was applied to all representative images. The glomerular basement membrane thickness was measured using Gaussian fits to line-profiles drawn perpendicularly across the membrane, n=50 measurements per condition. The full width at half-maximum is reported in Figure 3L.
For 3-D SIM, z-stacks of 19 planes of both channels (488 and 561 nm) were acquired from the stained kidney sections using the N-SIM super-resolution microscope (Nikon) equipped with a 100x silicone objective. The images were reconstructed into 3-D SIM images using NIS-Elements AR 5.30 (Nikon). The z-stacks were converted into a maximum intensity projection (MIP) followed by the automatized identification of the filtration slit length. The filtration slit density (FSD), meaning the length of the filtration slit per podocyte foot process area, was determined. This podocyte exact morphology measurement procedure (PEMP) has been described in detail previously (35). The FSD of 20 glomeruli was determined (n=5/group).
All graphs were set up using Prism 9 (GraphPad Software). Normality of the groups was tested using Shapiro-Wilk normality test. The groups were compared using one-way ANOVA corrected for multiple comparison by controlling the false discovery rate (FDR) using the method of Benjamini and Hochberg.
FDR-adjusted p-values of less than 0.05 were considered significant.

Assessment of Liver Aging
Fresh liver biopsies were preserved by snap freezing in OCT and stored at -80 C. Frozen sections were cut (8 µm thick) and used for staining as prescribed above. Hepatic lipids and triglycerides were determined by Oil Red O staining as previously described (91). To determine morphological changes present in aged liver, frozen liver sections (6µm) were stained for Collagen IV and the endothelial marker CD31/PECAM-1 as previously described (92). (1) Raw reads from fastq files were aligned to mm10 using the SubRead aligner (94). Gene-level read counts were obtained using htseq-count (95). Genes with less than 10 normalized reads summed across all samples were removed from further analysis. DESeq (96) was used to identify differentially expressed genes (DEGs), which were defined as genes with false discovery rate <0.05 and >2-fold change comparing young to aged. We additionally required that a DEG should have mean expression above 4 RPKM in either young or aged mice.
Genes were first ordered based on the pi score, a metric that combined p-value and fold change (98).
Genes with both a large increase in expression in aged podocytes and high statistical significance were at the top of the list; genes with both a large decrease in expression and high statistical significance were at the bottom of the list. Genes with moderate expression fold change and/or statistical significance were ranked in the middle. This ranked gene list was then used as input for GSEA.
(3) Additionally, the top Gene Ontology (GO) package (99) was used for GO enrichment analysis, based on Fisher's exact test. In order to visualize the interactions between genes within perturbed pathways, we mapped DEGs onto network diagrams from the KEGG pathway database (100) using the PathView tool (101).
(4) The VIPER (virtual inference of protein activity by enriched regulon analysis) method was used to identify potential master transcriptional regulators of podocyte aging as we described previously (42) Genes were sorted from the most down-regulated genes in aged podocytes to the most up-regulated genes in aged podocytes compared to young podocytes. If a significant fraction of a TF's positive targets were up-regulated, and its negative targets down-regulated in aged podocytes, this TF is inferred to be activated in aged podocytes (inactivated in young podocytes); if a significant fraction of a TF's positive targets are down-regulated and its negative targets are up-regulated in aged podocytes, the TF is inferred to be inactivated in aged podocytes (activated in young podocytes).

PD-1 Overexpression in Mouse Podocytes
The Lenti-X 293T Cell Line (Takara Bio USA, Inc., Mountain View, CA) was cultured according to

Gene Expression Analysis of Human Kidneys
Kidney tissue was obtained from the unaffected parts of kidneys removed from patients undergoing surgery at the University of Michigan and processed via the tissue procurement service of the Department of Pathology. Clinical data were obtained through the honest broker office of the University of Michigan as we have reported (102,103). Tissue was placed right away in RNAlater, micro-dissected into glomeruli and tubulo-interstitial fractions, and isolated RNA was used for gene expression analysis using Affymetrix Human Gene 2.1 ST Array (22, 103). This study was approved by the Institutional Review Board of the University of Michigan.

Injury Analysis of Human Kidney Biopsies
Young kidneys consisted of nephrectomy specimens from trauma victims under the age of 30 years, which demonstrated normal renal parenchyma. Aged kidney specimens from patients greater than 70 years of age with tubulointerstitial injuries (acute interstitial nephritis or acute tubular necrosis) were utilized. Focal segmental glomerulosclerosis (FSGS) specimens were generally of the not otherwise specified variant and all patients had nephrotic-range proteinuria. To determine if the clinical usage of immune checkpoint inhibitor therapy (ICPI) affected human podocytes, we queried the electronic health record to identify the ten most recent consecutive cases of patients with kidney biopsy and ICPI treatment at the University of Washington Medical Center. The kidney biopsies were examined closely for podocyte changes.

Statistical Analysis
Data are shown as the mean ± S.E.M. Student's t-test was applied for comparisons between groups.
Multiple groups were compared using one-way ANOVA with post hoc Tukey HSD test. P values <0.05 were considered statistically significant differences.

Supplemental Figure 1. Experimental Design and Physiological data for study animals. (A)
Schematic of study design. 4-month-old mice comprised the young group. The aged group of 21-monthold mice were randomized to the control group which received 8 weekly intraperitoneal (IP) injections of rat IgG2a (control), or the treatment group which received 8 weekly IP injections of anti-PD1 antibody. (B) For the duration of the study, aged animals did not lose weight with weekly injections of either the IgG2a control (red bars) or anti-PD1 antibody (blue bars) and weights were similar to the young group (black bar). (C-F) For the duration of the study, kidney function parameters, ACR (C), BUN (D), creatinine (E) and plasma suPAR levels (F) were measured. Yet, none changed significantly between young (4 months), aged IgG2a control (23 months), or anti-PD1 antibody (23 months) injected mice, with the exception of creatinine levels between the young and the aged IgG2a-injected mice. (G) Immunofluorescence staining for rat IgG2a confirmed that the injected aPD1ab reached the glomerular (dashed box) and tubular (white arrows) epithelium and that the binding patterns was similar to the staining pattern observed for PD1 expression by immunofluorescence. Similarly, the distribution in the liver also reflected the PD1 expression pattern in the liver (white arrow). No signal was detected in the control IgG2a antibody-injected kidney and liver.

Supplemental Figure 2. Glomerular endothelial cell density/injury and mesangial cell density. (A-D)
Representative images of double staining for the glomerular endothelial cell marker ERG (brown, nuclear) and collagen IV (blue, outlines glomeruli) and quantification thereof. Note that the agedependent decrease in endothelial cell density does not change with aPD1ab treatment. (E-G) Representative images of immunoperoxidase staining for the fenestra diaphragm protein plasmalemmal vesicle associated protein-1 (PV-1) (brown) was not detected in glomerular endothelial cells of young mice, but was increased in aged IgG2a injected mice and decreased in aPD1ab injected mice. Superscripted images show higher magnification of the glomeruli highlighted by dashed boxes.  (PD1, A), Cd274 (PD-L1, B) and Pdcd1lg2 (PD-L2, C) in non-podocytes and podocyte cell fractions of the kidney. In the non-podocyte fraction, aPD1ab lowered Pdcd1, but did not change levels of Cd274 or Pdcd1lg2 compared to IgG2a-injected mice. In podocytes, aPD1ab injection appeared to lower Pdcd1, Cd274 and Pdcd1lg2, compared to IgG2a-injected mice, but this did not reach statistical significance due to variation with the groups. (D) Gene expression data from the mRNA-seq experiment was mapped to PD1 target pathways in KEGG pathway database. Red rectangles denote up-regulated genes (aged/young expression ratio >1), blue rectangles denote downregulated genes (aged/young expression ratio <1); grey rectangles denote genes with no significant change. Edges represent interactions between genes/proteins. "+p" denotes phosphorylation. Sharp arrows indicate positive, while dashed arrows indicate negative regulation. Scale in upper right shows fold change by color hue. Note that the panel shows an overall significant decrease in genes downstream of PD1 in aged podocytes from mice injected with aPD1ab compared to control aged-matched mice given IgG2a. Figure 4. Gene expression data mapped to the tight junction pathway in the KEGG pathway database. Gene expression data of the mRNA-seq experiment was mapped to tight junction pathways in the KEGG pathway database. Red rectangles denote up-regulated genes (aged/young expression ratio >1), blue rectangles denote down-regulated genes (aged/young expression ratio <1); grey rectangles denote genes with no significant change. Edges represent interactions between genes/proteins. "+p" denotes phosphorylation. Sharp arrows indicate positive, while dashed arrows indicate negative regulation. Scale in upper right shows fold change by color. The panel shows an overall significant increase in genes for tight junctions in aged podocytes from mice injected with aPD1ab compared to control aged-matched mice given IgG2a. Phosphorylation mapped to the KEGG pathway database. Panel shows genes that are decreased (blue) and increased (red) in the oxidative phosphorylation pathway in aged mice injected with anti-PD1 antibody (aPD1ab) compared to control aged mice injected with the IgG2a. Scale in upper right shows with the degree of fold change indicated by color hue. Figure 8. Gene expression data mapped to the Glycolysis/Gluconeogenesis pathway in the KEGG pathway database. (A) Gene expression data was mapped to the Glycolysis/Gluconeogenesis Pathway in the KEGG pathway database. Red rectangles denote upregulated genes (aged/young expression ratio >1), blue rectangles denote down-regulated genes (aged/young expression ratio <1); grey rectangles denote genes with no significant change. Edges represent interactions between genes/proteins. "+p" denotes phosphorylation. Sharp arrows indicate positive, while dashed arrows indicate negative regulation. Scale in upper right shows fold change by color hue. The panel shows an overall significant increase in genes for to the Glycolysis/Gluconeogenesis Pathway in aged podocytes from mice injected with aPD1ab compared to control aged-matched mice given IgG2a. Figure 9. Gene expression data mapped to the Citrate Cycle pathway in the KEGG pathway database. (A) Gene expression data was mapped to the Citrate Cycle Pathway in the KEGG pathway database. Red rectangles denote up-regulated genes (aged/young expression ratio >1), blue rectangles denote down-regulated genes (aged/young expression ratio <1); grey rectangles denote genes with no significant change. Edges represent interactions between genes/proteins. "+p" denotes phosphorylation. Sharp arrows indicate positive, while dashed arrows indicate negative regulation. Scale in upper right shows fold change by color hue. The panel shows an overall significant increase in genes for to the Citrate Cycle Pathway in aged podocytes from mice injected with aPD1ab compared to control aged-matched mice given IgG2a. Figure 10. Tubulointerstitial Changes with aPD1ab injection. (A-D). Representative images of Collagen IV immunoperoxidase staining (brown) and their quantification thereof. Note that compared to young kidneys, Collagen IV staining was higher in the interstitium of aged IgG2a-injected mice, which was decreased by aPD1ab injection. (E-G) Representative images of Interleukin 17a (IL17a) immunofluorescence staining (green). Compared to young kidneys (E), IL17a staining was higher in the tubular epithelial cells in aged IgG2a-injected mice (F) and was decreased by aPD1ab injection (G).